Neurons within the respiratory network regulate and coordinate respiratory pump and airway muscle activation. As such, the level of pulmonary ventilation is determined by the balance of excitatory and inhibitory inputs to these neurons. It has long been assumed that the primary excitatory drive to breathe is from intracranial CO2- H+ chemoreceptors. However, severe hypoventilation and reduced central CO2-H+ chemosensitivity after carotid body denervation (CBD) suggest a major excitatory drive is provided by carotid chemoreceptor afferents. It is unknown how, despite the presence of the intact and highly-sensitive intracranial chemoreceptors, CBD leads to hypoventilation, and furthermore what mechanisms of plasticity govern the normalization of breathing two or more weeks after CBD. One proposed pathway (Res. Plan, Figure 1, page 1) for these excitatory carotid chemoreceptor effects is through second order solitary tract (NTS) neuronal projections to the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN). Phox2b-expressing (Phox2b+) pFRG/RTN neurons are hypothesized to: 1) receive and integrate multiple excitatory inputs, including those from the carotid bodies, and 2) provide the critical excitatory drive to pre-Bvtzinger complex (preBvtzC) respiratory rhythmogenic neurons during sleep;hence, dysfunction of Phox2b+ neurons underlies central and obstructive sleep apnea, and abnormal chemoreceptor and exerciseventilatory responses. An alternative hypothesis is that the carotid excitatory effect is through changes in neuromodulator inputs to brainstem respiratory neurons. We will test these and other hypotheses with the following Specific Aims: 1) Determine the effects on breathing in awake and asleep goats of destruction of Phox2b+ pFRG/RTN neurons. Hypothesis: Bilateral destruction of Phox2b+ pFRG/RTN neurons will cause: a) hypoventilation while awake which will be accentuated during NREM sleep by prolonged apneas, and b) attenuated ventilatory responses to hypercapnia, hypoxia, and exercise. Validation of these hypotheses will support the concept that the pFRG/RTN neurons provide critical excitatory input for breathing, particularly during sleep. 2) Determine whether CBD in goats induces central shifts in the balance between excitatory and inhibitory neuromodulation of the brainstem respiratory network. Hypothesis: After CBD when goats hypoventilate and CO2 sensitivity is reduced, the concentration of excitatory and inhibitory neuromodulators in effluent mock cerebrospinal fluid (mCSF) dialyzed through the preBvtzC will be decreased and increased respectively from baseline. Also after CBD, the ventilatory response to an injection of a glutamate receptor agonist into the preBvtzC will be reduced. Validation of these hypotheses will support the concept that tonic excitatory carotid activity affects neuromodulator-mediated excitability of respiratory rhythmogenic neurons. 3) Determine whether the observed time-dependent plasticity (recovery) after CBD is through upregulation of excitatory neuromodulatory mechanisms. Hypothesis: Two weeks after CBD in goats when they are no longer hypoventilating, the concentrations of excitatory and inhibitory neuromodulators in effluent mCSF dialyzed through the preBvtzC and the ventilatory response to a glutamate receptor agonist injection into the preBvtzC will be at or above normal. Furthermore, post-mortem immunohistochemistry will show increased percentage of neurons with receptors for excitatory neuromodulators in the preBvtzC. Validation of these hypotheses will be consistent with the concept that plasticity after CBD is due to upregulation of brainstem excitatory neuromodulatory mechanisms within the respiratory network. 4) Determine the effects on breathing in awake and sleeping goats of CBD after pFRG/RTN lesions. Hypothesis: When goats undergo CBD a month after lesioning the pFRG/RTN, the effects on breathing will be less than what normally occurs after CBD. Validation of this hypothesis will indicate the major pathway (Res. Plan, Figure 1, page 1) for central mediation of carotid afferent excitatory effect is through the pFRG/RTN.

Public Health Relevance

In millions of Americans during sleep, the cycling mechanism of breathing does not function properly or airways collapse resulting in central and obstructive sleep apnea respectively. The repeated apneas result in low O2 and high CO2 in the body which in many leads to arterial hypertension, strokes, heart attacks, daytime somnolence, dysphasia, and psychiatric disorders. These breathing disorders and the resultant diseases are prominent in the VA population leading to poor quality of life of many veterans. In the proposed studies on goats, we will eliminate part of the brain's system that regulates breathing which we hypothesize will result in disordered breathing during sleep. However, we expect that by a month later, breathing will have returned toward normal. Post mortem studies on brain cells will provide insight into how the brain recovers from major injury. Thus the present study will be another step toward improving care and management of veterans with sleep disordered breathing and also for military personal that had brain injury during combat.